Growth and division of cells require a cell cycle, that is, a regular sequence of processes. Two processes of the cell cycle are the accurate replication of DNA and the segregation of chromosomes to the two daughter cells during mitosis. When mitosis is not taking place, the cell is in an interphase period. Interphase is subdivided into three phases; a synthetic phase known as S-phase, when DNA synthesis takes place, and G1 and G2, gap phases that separate S-phase from mitosis. G1 is the gap after mitosis before DNA synthesis starts, and G2 is the gap after DNA synthesis is complete before mitosis and cell division. The mechanisms that insure alteration between S-phase and G-phases are critical to achieving cells of correct size and chromosome number. Most cells divide after replication of their chromosomes, and most cells replicate their chromosomes after completing cell division. Cells rely on checkpoints that link cell division processes and chromosome replication processes.
Checkpoint mechanisms guarantee that eukaryotic cells maintain genomic integrity during cell division by monitoring damaged or incompletely replicated. Such mechanisms ensure that cell cycle progression is stalled until aberrant DNA structures or replication intermediates can be eliminated. An ultimate target of these control mechanisms is the Cdc2-cyclin B complex, also known as maturation or M-phase promoting factor (MPF). During interphase, the Cdc2 subunit of MPF is down-regulated by inhibitory phosphorylations on its Thr-14 and Tyr-15 residues. A regulatory system containing two inhibitory kinases, Mytl and Weel, and a stimulatory phosphatase, Cdc25C, controls the activity of Cdc2 through reversible phosphorylation of these residues. Checkpoint mechanisms prevent the activating dephosphorylation of Cdc2 at the G2/M transition unless two accurate copies of the genome are available for transmission to daughter cells.
Genetic studies in yeast have identified proteins that are involved in sensing information damaged and/or incompletely replicated DNA and transmitting this information to effector molecules that interact directly with the cell cycle control machinery. In fission yeast, the sensor proteins are currently thought to include Rad1, Rad3, Rad9, Rad17, Rad26, Hus1, and Crb2/Rhp9. Effector proteins include the kinases Chk1 and Cds1 as well as 14-3-3 proteins such as Rad24. Similar pathways are found in budding yeast, and homologues of many of these proteins have been identified in higher eukaryotes, including humans, Xenopus, Drosophila, and mice. The most well established function of Chk1 in yeast and vertebrates is mediation of the binding of 14-3-3 proteins to Cdc25, which results in its cytoplasmic localization.
Phosphoinositide kinase relatives in fission yeast (Rad3), budding yeast (Mec1), and vertebrates (Atm and Atr) play an essential role in signaling the presence of damaged and/or unreplicated DNA to downstream regulators. For example, in fission yeast, Chk1 and Cds1 cannot function normally in the absence of Rad3. Similarly, Mec1 is a critical regulator of Rad53. a Cds1 homologue in budding yeast. In vertebrates, Atm is an upstream regulator of Chk2/Cds1. Atr is essential for genomic stability and early embryonic viability.
Despite these insights about certain components of checkpoint mechanisms, relatively little is known about how the Cds1 and Chk1 families respond to checkpoint signals. For example, in budding yeast, Rad53, a presumed target of Mec1, must bind to Rad9 to undergo phosphorylation and activation. Chk1 also becomes phosphorylated during checkpoint responses in various organisms. Although it is widely assumed that this phosphorylation leads to activation of Chk1, experimental proof of this possibility has not been provided. In fission yeast, both the phosphorylation of Chk1 and the ability of Chk1 to function in checkpoint control depend upon Crb2/Rhp9, a relative of budding yeast Rad9. Genetic and two-hybrid experiments have established a close relationship between Chk1 and Crb2/Rhp9, but a direct interaction between these proteins has not been reported.
In view of the importance of cell division and development, there is a need for determining further biochemical components of checkpoint mechanisms. This invention meets that need and provides methods of using such components.